The reductive half-reaction of two bifurcating electron-transferring flavoproteins: Evidence for changes in flavin reduction potentials mediated by specific conformational changes

• Published in: The Journal of biological chemistry 2022-06, Vol.298 (6), p.101927
• Main Authors: Vigil, Jr, Wayne, Tran, Jessica, Niks, Dimitri, Schut, Gerrit J, Ge, Xiaoxuan, Adams, Michael W W, Hille, Russ
• Format: Article
• Language: English
• Subjects: Electron Transport; Electron-Transferring Flavoproteins - chemistry; Electron-Transferring Flavoproteins - metabolism; Ferredoxins - metabolism; Flavin-Adenine Dinucleotide - chemistry; Flavin-Adenine Dinucleotide - metabolism; Flavins - metabolism; NAD - metabolism; Oxidation-Reduction; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Protein Structure, Tertiary
• ISSN: 1083-351X; 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2022.101927

The EtfAB components of two bifurcating flavoprotein systems, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from the bacterium Megasphaera elsdenii and the menaquinone-dependent NADH:ferredoxin oxidoreductase from the archaeon Pyrobaculum aerophilum, have been investigated. With both proteins, we find that removal of the electron-transferring flavin adenine dinucleotide (FAD) moiety from both proteins results in an uncrossing of the reduction potentials of the remaining bifurcating FAD; this significantly stabilizes the otherwise very unstable semiquinone state, which accumulates over the course of reductive titrations with sodium dithionite. Furthermore, reduction of both EtfABs depleted of their electron-transferring FAD by NADH was monophasic with a hyperbolic dependence of reaction rate on the concentration of NADH. On the other hand, NADH reduction of the replete proteins containing the electron-transferring FAD was multiphasic, consisting of a fast phase comparable to that seen with the depleted proteins followed by an intermediate phase that involves significant accumulation of FAD⋅ , again reflecting uncrossing of the half-potentials of the bifurcating FAD. This is then followed by a slow phase that represents the slow reduction of the electron-transferring FAD to FADH , with reduction of the now fully reoxidized bifurcating FAD by a second equivalent of NADH. We suggest that the crossing and uncrossing of the reduction half-potentials of the bifurcating FAD is due to specific conformational changes that have been structurally characterized.